We propose to use single-molecule spectroscopy imaging simultaneously with single-channel current recording to interrogate the dynamics in the conformational motions of a single ion-channel, to better understand the mechanism of channel function at the cell membranes. To meet a major methodology and technical need and challenge in the medical life sciences, the primary goal of our proposal is to solve a critical problem that holds understanding ion channel protein biological function and dynamics over the last four decades ever since the patch-clamp electrophysiological technique demonstrated. Specifically, we propose to conduct a systematic technical development and demonstration. Our project consists of three primary aims: (1) Achieve an experimental understanding of the sub-unit conformational changes that control the open-close activity of the NMDA receptor; (2) Resolve the conformational changes of the NMDA receptor in desensitization and inactivation; and (3) Demonstrate high time resolution for single-molecule ion channel dynamics studies. Our proposed technical approach, patch-clamp confocal single-molecule fluorescence imaging microscopy, will be applicable for a broad range of ion channel proteins and receptors in various cells, including all of the cellular systems that have been traditionally studied by conventional patch-clamp electric current measurements and proteins that can be probed by fluorescence. We will use the N-methyl-D-aspartate (NMDA) receptor in living cells as a model system to demonstrate our innovative physical technique and its unprecedented applications in life sciences and medical researchers. The dynamics of NMDA receptor mediated calcium currents is crucial to the normal function of the brain. The temporal behavior of NMDA receptor activity is regulated by the dynamic conformational changes of the protein in response to agonist binding and dissociation. Identifying the conformational motions of the receptor is critical for understanding the mechanisms of receptor function. Inhomogeneous conformational motions of the receptor control the activation, inactivation, desensitization and deactivation of the channel that in turn shape the calcium transients. PUBLIC HEALTH RELEVANCE: The kinetic behavior of the NMDA receptor plays a crucial role in the normal function of the brain. NMDA receptor mediates calcium current controls intracellular molecular cascades that determine the fate of synapses and cells. While the human health role of the NMDA receptor is important, our understanding of its dynamics and function is still obscured. Governed by the conformational dynamics of the protein, the precise and complex kinetic behavior of the channel is critical for the execution of cognitive functions and brain development. It is therefore of great importance to understand the conformational dynamics that underlie the NMDA receptor kinetic behavior. Abnormal activation of the NMDA receptor under pathological conditions leads to cell-death, and plays a role in the etiology of several neurodegenerative diseases. The NMDA receptor has been the target for the development of antagonists as therapeutic drugs. Analysis of the NMDA receptor is therefore highly relevant to public health.